The present invention relates to assay methods and kits for detecting nucleotide sequences in the nucleic acid (DNA or RNA) of a biological sample.
Nucleic acid assays based upon the specificity of hybridization have been described, e.g., in U.S. Pat. Nos. 4,358,535 to Falkow, et al (1982) and 4,486,539 to Ranki, et al (1984) and in PCT Application WO 84/02721 of Kohne (1984). Nucleic acid assays also providing specificity at restriction sites have been described, e.g., in U.S. Pat. No. 4,395,486 to Wilson, et al (1983) and in B.J. Conner, et al, Proc. Nat. Acad. Sci U.S.A., vol. 80, pp. 278-282 (1983). There is a continuing need for improvement in such assays, particularly with regard to specificity, ease of use, reduced assay time, increased sensitivity and reduction in background signal. A particular need exists for assays capable of clearly distinguishing closely related sequences such as the highly conserved ribosomal RNA (rRNA) strands present in high copy number in related organisms (e.g., the rRNA strands of E. coli. and of Salmonella typhimurium).
EP-A-123,513 of Mundy (Amersham International) (1984) describes a method of detecting a single nucleotide mutation. A target strand (typically immobilied) is hybridized with a labeled probe to form a hybrid double-stranded region up to the nucleotide adjacent to the site of the suspected mutation. Probe extension is then conducted in the presence of a single modified nucleotide precursor (e.g., a thionucleoside triphosphate) that will extend the probe by one nucleotide if one base is at the suspected site, but not at all if any other base is at the suspected site. The duplex is then subjected to digestion conditions that will digest probe that has not been extended, but that will not digest probe which has been extended by one modified nucleotide. Label (elsewhere on the probe) is then detected if still in a duplex (i.e., if not digested).